One example application is Total Internal Reflection Microscopy, which is a technique for observing samples illuminated by an evanescent wave. Total internal reflection occurs when a beam of light travelling through a very dense medium such as glass encounters an interface with a less dense medium such as air or water, at an angle to the normal which is greater than the critical angle for the interface. The critical angle for a glass/water interface is given by Fresnel's Law of Refraction as:θc=sin−1 (nwater/nglass)
At angles greater than the critical angle, when total internal reflection takes place, an electric field component of the light penetrates through the interface into the water as an evanescent wave. The evanescent wave has the same wavelength as the incident beam but penetrates only a very short distance into the water, typically no more than 1 μm. The evanescent wave decays exponentially from the interface into the water with a characteristic penetration depth dependent on the wavelength and angle of incidence of the totally internally reflected light.
In Total Internal Reflection Fluorescence Microscopy, fluorophores may be excited by the light in the evanescent field if they are close to the glass/water interface, but fluorophores further away in the bulk of the solution will not be excited. The result is that images with very low background fluorescence are obtained. FIG. 1 shows a typical instrument set up used in Total Internal Reflection Fluorescence Microscopy. A sample is placed such that it is located directly on the interface of the base of a light coupling optic or dispersion prism 1. Alternatively, a glass slide 2 may be optically matched to the prism, and the sample located on the base of the slide. Total internal reflection then occurs at the base of the slide. Typically, the objective lens 3 and external light source 4 are fixed in the lab frame and the sample on which the light coupling optic or prism is fixed is scanned in a plane perpendicular to the objective lens axis. The prism 1 therefore moves relative to the objective lens 3 and the light source 4. Conventionally a 45° or 60° dispersion prism is used, but to obtain light beams incident on the base of the prism at angles close to and greater than the critical angle, the light must usually be incident on the input face of the prism at off normal angles of incidence to achieve refraction of the beam at the air/glass interface. The deviation of the beam causes the reflection footprint at the base of the prism 1 to walk db as the prism is translated dx towards or away from the light source. In a limiting case light propagating parallel to the prism base will be refracted such that the footprint at the prism base moves equally and in the same direction as the prism (db/dx0). In this case the footprint moves dx in the lab frame and the illuminated area moves rapidly away from the imaging lens as the sample is scanned.
In imaging systems in which excitation or illumination of a sample is largely confined to a sample plane, such as in total internal reflection fluorescence microscopy (TIRFM), accurate excitation and imaging of material in this plane can be extremely sensitive to movements or irregularities of the plane. This is especially true in systems of high magnification, since a higher magnification generally results in a smaller field of view and depth of focus. If the sample is scanned, replaced or otherwise moved, adjustments need to be made to keep the image in focus. If the illumination beam is obliquely incident on the sample plane, as it is in TIRFM, irregularities or movements in the sample plane cause the intersection, or footprint of the beam in the plane to move laterally across the imaging area. This effect may be termed footprint misalignment, and results in the objective or imaging lens looking at a different part of the sample plane to that which is being illuminated.
Variations and irregularities may be present across a particular sample, causing footprint misalignment as the sample is scanned. Variations may also arise between consecutive samples, making an initial alignment of the illumination beam necessary when a new sample is loaded or set up.
Footprint misalignment has been found to be a particular problem in setting up a new sample for scanning with the TIRFM technique. In FIG. 2, the TIRFM arrangement of FIG. 1 is represented in section. An illumination laser beam 5 is transmitted through the prism 1, before passing through a layer of index matching fluid into the sample slide 2. The thickness of the slide in the drawing has been greatly exaggerated for clarity. The sample plane is defined by the lower surface of the slide. The angle of incidence of the beam onto the lower surface of the slide is sufficiently oblique that total internal refraction takes place, and none of the illumination beam propagates through the bottom of the slide 2. For a glass-water interface an angle of incidence of about 68° is generally appropriate for ensuring total internal reflection of all components of the beam.
In the unaligned arrangement shown in FIG. 2, the focal plane 10 of the objective lens 3 lies within the slide 2, and the illumination footprint 12 lies to the left of the field of view of the objective lens 3. Approximate focus of the objective on the sample 14 can be achieved by, for example, observing the geometry of a drop of immersion oil between the objective lens 3 and a cover slip placed over the sample 14, thus bringing the focal plane 10 roughly into coincidence with the sample 14. Approximate alignment of the footprint 12 with a region of interest of the sample surface being imaged using the objective lens can then be achieved by adjusting the illumination laser beam 5 and observing the scattered light until a high contrast background is observed, as long as the focus is not too far from correct. This high contrast background originates from point defects and irregularities in the surface and may be described as a grainy image, typically including bright circular rings which may consist of intermittent bright and dark rings (“airy discs”) when slightly defocussed or a bright point when in focus. Finding and correctly identifying this grainy image is difficult. If the footprint 12 is too far from the objective lens field of view, which may be very small, a largely blank, or at least less grainy image will result. If a bubble is present in either the immersion oil or index matching fluid or if light reflects from the rim of the objective lens aperture 3 then the image may become swamped with scattered light.
When a grainy image has been found, the objective lens 3 is translated towards or away from the sample 14 until a scratch or point defect on the slide-sample interface comes into focus, at the same time adjusting the footprint to complete the alignment. This process is often hampered by the presence of strong scatter, and is made more difficult because the focus in a first dimension and the footprint position in the two other dimensions need to be adjusted at the same time. The process becomes particularly difficult at high magnifications and correspondingly small depths of focus and small fields of view. These set-up difficulties present significant obstacles when designing TIRFM or similar systems which are suitable for automated or semi-automated high throughput and/or scanning applications. When the sample is scanned, irregularities in the sample surface, even if compensated for using an autofocus arrangement, can still result in footprint misalignment.